Synthesis of an Analog of the Thyroid Hormone-binding Protein
Transthyretin via Regioselective Chemical Ligation*
Jackie A.
Wilce
,
Stephen G.
Love§,
Samantha J.
Richardson¶,
Paul F.
Alewood§, and
David J.
Craik§
From the Department of Biochemistry/Chemistry,
University of Western Australia, Nedlands Western Australia 6907, Australia, the § Institute for Molecular Bioscience,
University of Queensland, Brisbane, Queensland 4072, Australia, and the
¶ Department of Biochemistry and Molecular Biology, University of
Melbourne, Parkville, Victoria 3010, Australia
Transthyretin is an essential protein responsible
for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic
polyneuropathy and senile systemic amyloidosis. Its folding
properties and stabilization by ligands are of current interest due to
their importance in understanding and combating these diseases. Here we
report the solid phase synthesis of the monomeric unit of a
transthyretin analog (equivalent to 127 amino acids) using
t-Boc chemistry and peptide ligation and its folding to
form a functional 54-kDa tetramer. The monomeric unit of the protein
was chemically synthesized in three parts (positions 1-51, 54-99, and
102-127) and ligated using a chemoselective thioether ligation
chemistry. The synthetic protein was folded and assembled to a
tetrameric structure in the presence of transthyretin's native ligand,
thyroxine, as shown by gel filtration chromatography, native gel
electrophoresis, transthyretin antibody recognition, and thyroid
hormone binding. Other folding products included a high molecular
weight aggregate as well as a transient dimeric species. This
represents one of the largest macromolecules chemically synthesized to
date and demonstrates the potential of protein chemical synthesis for
investigations of protein-ligand interactions.
*
This work was supported by a grant from the Australian
Research Council (to D. J. C. and J. A. W.). The Institute for
Molecular Bioscience is a Special Research Center of the Australian
Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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